Internalization of serratial protease into cells as an enzyme-inhibitor complex with alpha 2-macroglobulin and regeneration of protease activity and cytotoxicity

Extracellular serratial protease (56,000 Da) is known to be cytotoxic. Fluorescein isothiocyanate-labeled protease was found to form a complex with human alpha 2-macroglobulin (alpha 2M), and this enzyme-inhibitor complex was purified. The protease was found to be internalized by fibroblasts in culture as a complex with alpha 2M, which resulted in cell destruction. Regeneration of enzyme activity was confirmed in cells after 2-3 h of incubation. Chicken egg-white ovomacroglobulin, a homolog of human alpha 2M, formed a complex with this enzyme similarly and more tightly but failed to exhibit protease activity, cytotoxicity, and internalization into cells.     

Some properties of partially purified preparations of cAMP phosphodiesterase from beef heart]

Beef heart cAMP phosphodiesterase (EC 3.1.4.17) was isolated and partially purified using fractionation by ammonium sulfate and gel filtration on the columns with Sephadex G-200 and Sepharose 6B. This method allowed to preserve the enzyme binding to the low-molecular weight thermostable protein regulator of the phosphodiesterase activity. The enzyme preparation was purified 130--180-fold as compared to the original homogenate. The pH-dependence of the enzyme activity in the imidazole and tris -- buffers for the fraction with maximal activity was carried out. The kinetic analysis of this fraction revealed an abnormal kinetic behaviour with two Km values. The enzyme is represented by four forms differing in their molecular weights and possessing different capacity for activation by Ca2+ and protein regulator. No activation was observed in the forms with higher molecular weights, whereas the activity of the forms with lower molecular weights depended on the presence of Ca2+ and protein regulator. It is assumed that some of the above-described forms are capable of interconversions.     

Pyrimidine reducing enzymes of rat liver.

Dihydrouracil dehydrogenase (NADP+) (EC 1.3.1.2) was partially purified from the cytosol fraction of rat liver and fractionated by disc gel electrophoresis. A major and minor band were visualized by staining for enzyme activity. The substrate specificity of these bands was investigated. It was found that both bands were two to three times more active with dihydrothymine as substrate than with dihydrouracil in the presence of NADP+ and the optimum pH of 7.4. Mitochondrial fractions containing most of the NADH-dependent uracil reductase of rat liver cells were fractionated by centrifugation in sucrose density gradients. Two procedures involving linear or discontinuous gradients were used. By both, good separation of NADH- and NADPH- dependent reductases was achieved. Marker enzyme studies supported the view that the NADH-dependent enzyme is located principally in mitochondria whereas the NADPH-dependent enzyme is mainly in plasma and endoplasmic reticulum membranes. For the NADH-dependent reductase the apparent Km for thymine at pH 7.4 was 1.39 times that found for uracil whereas for the NADPH-dependent enzyme the apparent Km values were similar for the two substrates at this pH. Dihydrouracil was the principal product isolated by paper chromatography from the reaction mixture containing a partially purified fraction of mitochondria, uracil and NADH at pH 7.4. This fraction also catalyzed the formation of radioactive carbon dioxide from [2-14C]uracil. The proportion of CO2 formed by the mitochondria was about 10% of that formed by the original homogenate.     

Characteristics of solubilized human-somatotropin-binding protein from the liver of pregnant rabbits.

A specific binding site for somatotropin was solubilized by 1% (v/v) Triton X-100 from a crude particulate membrane fraction of pregnant rabbit liver, partially purified and characterized. The solubilized binding site retained many of the characteristics observed in the original particulate fraction, indicating that extraction of the binding site with Triton X-100 does not cause any major changes in its properties. The binding of human 125I-labelled-somatotropin to the solubilized binding site is a saturable and reversible process, depending on temperature, incubation time, pH and ionic environment. Analysis of the kinetic data revealed a finite number of binding sites with an affinity constant of 0.32 x 10(10)M-1. The binding activity for human 125I-labelled-somatotropin was adsorbed to a concanavalin-A-Sepharose column and was dissociated from the column with alpha-methyl-D-glucoside, suggesting that the binding protein may be a glycoprotein. Using affinity chromatography on concanavalin-A-Sepharose, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B, the binding protein was purified 1000-4000-fold from the original liver homogenate. When the partially purified preparation was chromatographed on Sepharose 6B, the binding protein eluted as a molecule with an apparent molecular weight of 200000, with a Stokes' radius of 4.9 nm. Sucrose-density-gradient centrifugation of the preparation showed that the sedimentation coefficient of the binding protein was 7.2S. Isoelectric focusing experiments revealed that a major part of the protein has an acidic pI (4.2-4.5). Exposure of the protein to trypsin decreased the binding activity for human 125I-labelled-somatotropin or bovine 125I-labelled-somatotropin, whereas ribonuclease, deoxyribonuclease, phospholipase C or neuraminidase had little or no effect.     

Purification and characterization of activated human erythrocyte prolidase

Prolidase (E.C. 3.4.13.9) has been purified 7500-fold to homogeneity from human erythrocytes in a Mn2+-activated form using conventional and fast protein liquid chromatography columns. The procedure includes a 1-h incubation of the crude hemolysate at 50 degrees C with 1 mM MnCl2. Following this novel step, prolidase retains full activity, obviating the requirement for preincubation of each enzyme fraction with Mn2+ prior to assay. Preincubation with MnCl2 does not change the isoelectric point of the enzyme. The molecular weight of the purified enzyme was 58,000 when measured by SDS-PAGE. Western blotting, using rabbit antibody raised to human kidney prolidase, with partially purified erythrocyte enzyme revealed a cross-reacting band at Mr 58,000.     

Interaction of model discoidal complexes of phosphatidylcholine and apolipoprotein A-I with plasma components. Physical and chemical properties of the transformed complexes

Conversion of model discoidal complexes of egg yolk phosphatidylcholine and apolipoprotein A-I, upon interaction with a source of lecithin:cholesterol acyltransferase (plasma d greater than or equal to 1.21 g/ml fraction or partially purified enzyme) and with different sources of substrate unesterified cholesterol (LDL, VLDL or cholesterol incorporated into complexes), was investigated by gradient gel electrophoresis, gel filtration, equilibrium density gradient ultracentrifugation, electron microscopy and chemical analysis. When the incubation mixture contained an inhibitor of lecithin:cholesterol acyltransferase, discoidal complexes with mean long dimension of approximately 10.5 +/- 1.9 nm were converted (within 1 h) predominantly to small round particles and were partially depleted of their phospholipid content. Upon electrophoresis the small particles showed peak maxima within the migration intervals of the human plasma ( HDL3b ) gge and ( HDL3c ) gge subpopulations with associated particle size ranges of 7.8-8.2 and 7.2-7.8 nm, respectively. Within 1 h, in the presence of activated enzyme, the complexes were again converted in major part to the small particles. However, further incubation resulted in an apparent single-step conversion to a larger major product with peak maximum occurring within the migration intervals of the ( HDL2a ) gge and the ( HDL3a ) gge subpopulations (particle size ranges 8.8-9.8 and 8.2-8.8 nm, respectively). Formation of an apolar core was indicated by detection of cholesteryl esters in the conversion product. The form in which the substrate unesterified cholesterol was introduced did not markedly influence the size properties of the final conversion product. With VLDL as source of substrate, considerable incorporation of triacylglycerol occurred in company with a lower level of cholesteryl esters, suggesting transfer of these lipids during formation of the apolar core. Incubation of complexes with a partially purified (3000-fold) preparation of lecithin:cholesterol acyltransferase yielded a product similar in properties to that when the d greater than or equal to 1.21 g/ml fraction was used. Our model discoidal complexes and their conversion products exhibit properties very similar to those of potential precursors to HDL as well as of mature HDL particles. Their further investigation shows promise of providing detailed insight into the possible origin and heterogeneity of human plasma HDL.     

Evidence for the presence of diacylglycerol kinase in rat brain myelin

The previous demonstration that incubation of brain slices with [³²P]phosphate brings about rapid tabeling of phosphatidic acid in myelin suggests that the enzyme involved should be present in this specialized membrane. DAG kinase (ATP:1,2-diacyglycerol 3-phosphotransferase, E.C. 2.7.1.107) is present in rat brain homogenate at a specific activity of 2.5 nmol phosphatidic acid formed/min/mg protein, while highly purified myelin had a much lower specific activity (0.29 nmol/min/mg protein). Nevertheless, the enzyme appears to be intrinsic to this membrane since it can not be removed by washing with a variety of detergents or chelating agents, and it could not be accounted for as contamination by another subcellular fraction. Production of endogenous, membrane-associated, diacylglycerol (DAG) by PLC (phospholipase C) treatment brought about translocation from soluble to particulate fractions, including myelin. Another level of control of activity involves inactivation by phosphorylation; a 10 min incubation of brain homogenate with ATP resulted in a large decrease in DAG kinase activity in soluble, particulate and myelin fractions.     

Insulin degradation XVIII. On the regulation of glutathione-insulin transhydrogenase in the hyperglycemic obese (ob/ob) mouse

The occureence of insulin-degrading activity in the liver of the obese hyperglycemic mouse (ob/ob) and its litter mate has been studied. The trichloroacetic acid-soluble product formed from insulin upon incubation with liver homogenate was identified as the A chain of insulin. In Ouchterlony double-diffusion experiments with antibody to purified rat liver glutathione-insulin transhydrogenase, mouse liver homogenate and the microsomal fraction each gave a single precipitation band of identity with the purified rat liver enzyme. These results indicate that the insulin-degrading activity preseny in the mouse liver is, in fact, glutathione-insulin transhydrogenase. Subcellular distribution studies of glutathione-insulin transhydrogenase and marker enzymes indicate that the transhydrogenase is located primarily in the microsomal fraction of mouse liver homogenate.The ob/ob mouse, which is a genetic mutant characterized by obesity, hyper-insulinism and resistance to the hypoglycemic action of insulin, contains hepatic glutathione-insulin transhydrogenase activity (per mg microsomal protein) markedly higher (40–60%) than its lean litter mates. However, a major portion of the increased hepatic enzyme in the ob/ob mouse occurs in a latent state; the increased amount of enzyme either is unavailable or is nonfunctional, although the ob/ob mouse still contains more of the functional form than the lean mouse. Thus, the results are consistent with the suggestion that the hepatic glutathione-insulin transhydrogenase is probably under a feedback control by circulating insulin.     

Topography, purification and characterization of thyroidal NAD+ glycohydrolase

Subcellular fractionation of bovine thyroid tissue by differential pelleting and isopycnic gradient centrifugation in a zonal rotor indicated that NAD(+) glycohydrolase is predominantly located and rather uniformly distributed in the plasma membrane. Comparison of NAD(+) glycohydrolase activities of intact thyroid tissue slices, functional rat thyroid cells in culture (FRT(l)) and their respective homogenates indicated that most if not all of the enzyme (catalytic site) is accessible to extracellular NAD(+). The reaction product nicotinamide was predominantly recovered from the extracellular medium. The diazonium salt of sulphanilic acid, not penetrating into intact cells, was able to decrease the activity of intact thyroid tissue slices to the same extent as in the homogenate. Under the same conditions this reagent almost completely abolished NAD(+) glycohydrolase activity associated with intact thyroid cells in culture. The triazine dye Cibacron Blue F3GA and its high-M(r) derivative Blue Dextran respectively completely eliminated or caused a severe depression in the NAD(+) glycohydrolase activity of FRT(l) cells. The enzyme could be readily solubilized from bovine thyroid membranes by detergent extraction, and was further purified by gel filtration and affinity chromatography on Blue Sepharose CL-6B. The overall procedure resulted in a 1940-fold purification (specific activity 77.6mumol of nicotinamide released/h per mg). The purified enzyme displays a K(m) of 0.40mm for beta-NAD(+), a broad pH optimum around pH7.2 (0.1 m-potassium phosphate buffer) and an apparent M(r) of 120000. Nicotinamide is an inhibitor (K(i) 1.9mm) of the non-competitive type. The second reaction product ADP-ribose acts as a competitive inhibitor (K(i) 2.7mm). The purified enzyme splits beta-NAD(+), beta-NADP(+), beta-NADH and alpha-NAD(+) at rates in the relative proportions 1:0.75:<0.02:<0.02 and exhibits transglycosidase (pyridine-base exchange) activity. Anionic phospholipids such as phosphatidylinositol and phosphatidylserine inhibit the partially purified enzyme. A stimulating effect was observed upon the addition of histones.     

Lectin-binding domains on laminin

Chicken erythrocytes have been found to have at least two kinds of phospholipase A2. The first is a soluble enzyme from the cytosole fraction and has no calcium sensitivity. The second can be extracted from the plasma membrane fraction with the nonionic detergent Triton X-100. In this study the membrane-bound enzyme was partially purified by affinity chromatography on phosphatidylcholine-Sepharose, and its specific activity was increased 1100-fold compared with that of the cell homogenate without nuclei. It has an optimum pH of 8.5 and required calcium for maximum activity. It showed the specificity for both phosphatidylcholine and phosphatidylethanolamine, but reacted preferentially on the former substrate. Analysis by concanavalin A-Sepharose affinity chromatography revealed that the membrane-bound phospholipase A2 was retained on the resin and could be eluted specifically with a haptenic sugar, methyl alpha-D-mannopyranoside. The enzyme seems to be either a concanavalin A-binding glycoprotein or a part of a complex with certain concanavalin A-binding glycoproteins.     

Insulin degradation. XVIII. On the regulation of glutathione-insulin transhydrogenase in the hyperglycemic obese (ob/ob) mouse.

The occurrence of insulin-degrading activity in the liver of the obese hyperglycemic mouse (ob/ob) and its litter mate has been studied. The trichloroacetic acid-soluble product formed from insulin upon incubation with liver homogenate was identified as the A chain of insulin. In Ouchterlony double-diffusion experiments with antibody to purified rat liver glutathione-insulin transhydrogenase, mouse liver homogenate and the microsomal fraction each gave a single precipitation band of identity with the purified rat liver enzyme. These results indicate that the insulin-degrading activity present in the mouse liver is, in fact, glutathione-insulin transhydrogenase. Subcellular distribution studies of glutathione-insulin transhydrogenase and marker enzymes indicate that the transhydrogenase is located primarily in the microsomal fraction of mouse liver homogenate. The ob/ob mouse, which is a genetic mutant characterized by obesity, hyperinsulinism and resistance to the hypoglycemic action of insulin, contains hepatic glutathione-insulin transhydrogenase activity (per mg microsomal protein) markedly higher (40--60%) than its lean litter mates. However, a major portion of the increased hepatic enzyme in the ob/ob mouse occurs in a latent state; the increased amount of enzyme either is unavailable or is nonfunctional, although the ob/ob mouse still contains more of the functional form than the lean mouse. Thus, the results are consistent with the suggestion that the hepatic glutathione-insulin transhydrogenase is probably under a feedback control by circulating insulin.     

Dissociation of human choriogonadotropin from the pseudopregnant rat ovary as a complex to a receptor fragment during perifusion and incubation

Pseudopregnant rats were injected intravenously with radioactively-labelled human choriogonadotropin (CG). The animals were killed 2 h after the injection and the ovaries, liver and kidney were subjected to perifusion. Radioactivity was released from the ovaries at an increasing rate during perifusion, mainly in a complex form with a molecular size between human CG and the solubilized receptor-human CG complex. The increase in the rate of radioactivity release was inhibited by N-ethylmaleimide and CuCl2, but not by MgCl2, Trasylol, N alpha-tosyl-L phenylalanine chloromethyl ketone or N alpha-p-tosyl-L-lysine and CuCl2 chloromethyl ketone. Intact hormone dissociation from the complex at pH 3. After perifusion the ovarian tissue radioactivity only as receptor-hormone complexes. Only free radioiodine released from the control tissues, liver and kidney during perifusion. The low molecular weight hormone complex was also released from a homogenate of pseudopregnant rat ovaries prelabelled in vivo with radioactivity-labelled human CG during incubation in a hypotonic medium. The release of this complex was likewise inhibited by alkylating agents and heavy metals, and intact hormone dissociated from the complex at pH 3. A similar human CG complex was released also from purified receptor-human CG complex during incubation with ovarian homogenate, and presence of N-ethylmaleimide or use of heat inactivated ovarian homogenate inhibited this process. The present results indicate that the in vivo bound human CG sheds from the luteal tissue in perifusion and incubation as a low molecular weight complex. This may be a facet in the processing and elimination of occupied LH receptors from the ovary.     

Relationship of ATP Turnover, Polyphosphoinositide Metabolism, and Protein Phosphorylation in Sciatic Nerve and Derived Peripheral Myelin Subfractions from Normal and Streptozotocin Diabetic Rats

Sciatic nerve from streptozotocin-induced diabetic rats has previously been shown to incorporate more 32P into phosphatidylinositol-4,5-bisphosphate (PIP2) and the principal myelin proteins than normal nerve. In the present study, labeling of ATP and PIP2 was compared. Using nerve segments, [gamma-32P]ATP specific activity reached a plateau after incubation for 4 h with [32P]orthophosphate, whereas the specific activity of [32P]PIP2 rose much more slowly and was still increasing after 8 h. The rate of disappearance of radioactivity from prelabeled ATP was biphasic, with 75% being lost within 30 min and the remainder declining much more slowly for several hours thereafter. In contrast, no decrease in prelabeled PIP2 radioactivity could be detected for up to 4 h. The kinetics of ATP metabolism were not appreciably different for normal and diabetic nerve. However, after incubation with [32P]orthophosphate for 2 h, the specific activity of PIP2 was 50-120% higher in diabetic nerve. This phenomenon, therefore, cannot be ascribed to altered specific activity of the ATP precursor pool. Greater labeling of PIP2 in 32P-labeled diabetic nerve was present in purified myelin isolated using a simple discontinuous sucrose density gradient, but not in a "nonmyelin" fraction. When nerve homogenate was fractionated on a more complex gradient, three myelin-enriched subfractions were obtained which were heterogeneous as judged by morphological appearance, protein profile, and lipid metabolic activity. The proportion of total lipid radioactivity accounted for by PIP2 was elevated in all the subfractions relative to the homogenate. As compared to myelin subfractions from normal nerve, an increased percentage of 32P in PIP2 was obtained only in the major myelin subfraction from diabetic nerve. The phosphorylation of P0 relative to the other myelin proteins was also enhanced in this subfraction in nerve from diabetic animals.     

Solubilization of membrane-bound acid alpha-glucosidase by proteolysis

The membrane-bound acid alpha-glucosidase was purified partially (400-fold) from human placenta by solubilization with trypsin, concanavalin A-Sepharose chromatography, Ultrogel AcA-34 gel filtration, and Sephadex G-100 affinity chromatography. Two molecular forms of the enzyme were found in the final preparation of the purified enzyme. They were identical in molecular weight with a precursor (110 kDa) and an early intermediate form (105 kDa) of this enzyme. Also direct incubation of the membrane fraction without trypsin resulted in a release mainly of the 105 kDa form, which was inhibited by N-ethylmaleimide, but not by leupeptin, pepstatin or phenylmethylsulfonylfluoride. It was concluded that the precursor of acid alpha-glucosidase is an intrinsic membrane protein, which is transported into lysosomes after solubilization by proteolysis.     

Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain

Aspartate N-acetyltransferase (Asp-NAT; EC 2.3.1.17) activity was found in highly purified intact mitochondria prepared by Percoll gradient centrifugation as well as in the three subfractions obtained after the sucrose density gradient centrifugation of Percoll purified mitochondria; citrate synthase was used as a marker enzyme for mitochondria. The proportion of recoverable activities of Asp-NAT and citrate synthase were comparable in mitochondrial and synaptosomal fractions but not in the fraction containing myelin. Asp-NAT was solubilized from the pellet of the rat brain homogenate (26 000 g for 1 h) for the recovery of maximum activity and partially purified using three protein separation methods: DEAE anion exchange chromatography, continuous elution native gel electrophoresis and size-exclusion high performance liquid chromatography. Asp-NAT activity and the optical density pattern of the eluted protein from size-exclusion column indicated a single large protein (approximately 670 kDa), which on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed at least 10 bands indicative of an enzyme complex. This seemingly multi-subunit complex Asp-NAT was stable towards ionic perturbations but vulnerable to hydrophobic perturbation; almost 95% of activity was lost after 10 mm 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) treatment followed by size-exclusion chromatography. Asp-NAT showed an order of magnitude difference in Km between l-aspartate (l-Asp, approximately 0.5 mm) and acetyl CoA (approximately 0.05 mm). Asp-NAT showed high specificity towards l-Asp with 3% or less activity towards l-Glu, l-Asn, l-Gln and Asp-Glu. A model on the integral involvement of NAA synthesis in the energetics of neuronal mitochondria is proposed.     

Changes in the proportion of acetyl-CoA carboxylase in the active form in rat liver. Effect of starvation, lactation and weaning

1. The activity of acetyl-CoA carboxylase (EC 6.4.1.2) in extracts of freeze-clamped liver samples from fed or 24 h-starved virgin, pregnant, lactating and weaned rats was measured (i) immediately after preparation of extracts (;I activity'), (ii) after incubation of extracts with partially purified preparations of either rabbit muscle protein phosphatase 1 [Antoniw, Nimmo, Yeaman & Cohen (1977) Biochem. J.162, 423-433] or rabbit liver phosphatase [Brandt, Capulong & Lee (1975) J. Biol. Chem.250, 8038-8044] (;A activity') and (iii) after incubation with 20mm-potassium citrate before or after incubation with phosphatases (;C activity'). 2. Incubation of liver extracts at 30 degrees C without any additions resulted in activation of acetyl-CoA carboxylase that was shown to be due to dephosphorylation of the enzyme by endogenous protein phosphatase activity. This latter activity was not stimulated by Ca(2+) and/or Mg(2+) but was stimulated by 1 mm-Mn(2+). Incubation of extracts with either of the partially purified phosphatases (0.2-0.5 unit) resulted in faster dephosphorylation and activation. The activity achieved after incubation with either of the exogenously added phosphatases was similar. 3. The A and C activities increased during late pregnancy, were lower than in the virgin rat liver during early lactation and increased by 2-fold in liver of mid-lactating rats. Weaning of mid-lactating rats for 24 h resulted in no change in A and C activities but after 48 h weaning they were significantly lower than those in livers from suckled mothers. 4. The I activity followed a similar pattern of changes as the A and C activities during pregnancy and lactation such that, although the I/A and I/C activity ratios tended to be lower during late pregnancy and early lactation, there were no significant changes in I/A and I/C ratios between lactating and virgin animals. However, these ratios were significantly higher in liver from fed 24 h-weaned animals. 5. Starvation (24 h) resulted in a marked decrease in I activity for all animals studied except early-lactating rats. This was due to a combination of a decrease in the concentration of acetyl-CoA carboxylase in liver of starved animals (A and C activities) and a decrease in the fraction of the enzyme in the active form (lower I/C and I/A ratios). The relative importance of the two forms of regulation in mediating the starvation-induced fall in I activity was about equal in livers of virgin, pregnant and lactating animals. However, the decrease in I/A and I/C ratios was of dominating importance in livers of weaned animals. The A/C activity ratios were the same for livers from all animals studied. 6. The maximal activity of fatty acid synthase was also measured in livers and was highly and positively correlated with the A and C activities of acetyl-CoA carboxylase, suggesting that the concentrations of the two enzymes in the liver were controlled coordinately. 7. It is suggested that the lack of correlation between plasma insulin levels and rates of lipogenesis in the transition from the virgin to the lactating state may be explained by different effects of insulin and prolactin on the concentration of acetyl-CoA carboxylase in the liver and on the fraction of the enzyme in the active form.     

Change in phosvitin kinase activity during early development of the sea urchin

Protein kinase, which phosphorylated phosvitin at the expense of ATP but did not phosphorylate casein, protamine, and histone mixture, was obtained by DEAE-cellulose column chromatography of the extract from the embryos of the sea urchin, Strongylocentrotus intermedius. This enzyme, partially purified by DEAE-cellulose column, reversibly catalyzed the reaction of phosvitin phosphorylation. This indicates that the sea urchin embryos contain phosvitin kinase. Phosvitin kinase in sea urchin embryos is somewhat different from that found in the other types of cells, which are able to phosphorylate casein as well as phosvitin. In unfertilized eggs, the activity of this enzyme was found only in the supernatant fraction obtained by centrifuging the homogenate at 10,000g for 20 min. The activity in the embryos at the swimming and the mesenchyme blastula stage was higher than in unfertilized eggs, and was localized in the sedimentable fraction obtained by centrifuging the homogenate of the embryos at 10,000g for 20 min. The highest activity of phosvitin kinase was observed in the embryos at the mesenchyme blastula stage, and the enzyme activity became quite low at the late gastrula stage. The activity and the intracellular distribution of phosvitin kinase changed during the development. The enzyme in this sedimentable fraction was not solubilized with 1% Triton X-100 but was extracted by 1 M NaCl.     

AF64A: An Active Site Directed Irreversible Inhibitor of Choline Acetyltransferase

Ethylcholine mustard aziridinium ion (AF64A, MEChMAz) has been proposed as a cholinergic neuron-specific neurotoxin. We report that in further studies on its mechanism of action incubation of the cholinergic neuroblastoma X glioma cell line, NG-108-15, with 100 microM AF64A resulted in a rapid decrease in cellular choline acetyltransferase (ChAT) activity which preceded cytotoxicity. Thus, a 60-85% decrease in ChAT activity was measured within 5 h of AF64A exposure, whereas cell lysis (measured as the release of the cytosolic enzyme lactate dehydrogenase into the medium) did not become apparent until 18 h of AF64A exposure. This led us to examine the effects of AF64A on partially purified ChAT. We report a concentration- and time-dependent inhibition of partially purified ChAT by AF64A that could not be reversed by dialysis but could be prevented by coincubation of the enzyme and AF64A with choline but not with acetyl-coenzyme A. We present kinetic evidence that choline and AF64A compete for the same site on the enzyme. In addition, thiosulfate, which inactivates the aziridinium ion, eliminated AF64A's capacity to inhibit the enzyme. AF64A also irreversibly inhibited partially purified choline kinase and acetylcholinesterase but not lactate dehydrogenase, alcohol dehydrogenase, carboxypeptidase A, or chymotrypsinogen, enzymes that do not use choline as a substrate or product. Thus, the data suggest that AF64A acts as an irreversible active site directed inhibitor of ChAT and possibly other enzymes recognizing choline.     

A new mechanism for regulation of tyrosine 3-monooxygenase by end product and cyclic AMP-dependent protein kinase

Tyrosine 3-monooxygenase activity of the crude extract from rat striatum had a sharp pH optimum at pH 5.4 and showed almost no activity at or above pH 7. When the crude extract was partially purified by pH precipitation and chromatography on DEAE-cellulose, the enzyme showed a high activity in the pH range of 5.8 to 7.4. Incubation of the partially purified enzyme with catecholamines such as dopamine, norepinephrine, and epinephrine resulted in a remarkable decrease in the enzyme activity, as assayed at a neutral pH. This suppression of the enzyme activity by catecholamines differed from the well-known feedback inhibition which is competitive with respect to the pterin cofactor; the former occurred at a very much lower concentration of catecholamines even in the presence of a near-saturating concentration of a pterin cofactor, and the former was a time-dependent reaction. The enzyme, the activity of which had been suppressed by the incubation with dopamine, was remarkably activated by the incubation with the catalytic subunit of cyclic AMP-dependent protein kinase in the presence of an ATP-generating system. These results suggest that the activity of tyrosine 3-monooxygenase may be suppressed by its end products in a normal state and it may be stimulated by cyclic AMP-dependent protein kinase as occasion demands.     

Heparan sulphate sulphotransferase. Properties of an enzyme from ox lung

A heparan sulphate sulphotransferase was partially purified from an ox lung homogenate by (NH(4))(2)SO(4) precipitation. Various glycosaminoglycans were assayed as sulphate acceptors with this enzyme. The highest acceptor activity was obtained with desulphated heparin and heparan sulphate, which indicates that sulphate transfer may be to free amino groups of the substrate. Some heparan sulphate was (35)S-labelled by incubation with the enzyme and re-isolated. On treatment of this heparan [(35)S]sulphate with nitrous acid and separation of the degradation products on Sephadex G-15, a major peak of radioactivity was obtained, and identified as [(35)S]sulphate by high-voltage electrophoresis at pH5.3. The [(35)S]sulphate is believed to be derived from N-[(35)S]sulphated groups of heparan [(35)S]-sulphate. That the ox lung preparation contained an N-sulphotransferase was confirmed by the isolation of 2-deoxy-2-[(35)S]sulphoamino-d-glucose as the major product from the flavobacterial degradation of heparan [(35)S]sulphate.